Potential Cytotoxicity of Methionine Aminopeptidase Inhibitors on Mammalian Cells

Abstract

Tuberculosis (TB) is a disease of the lung caused by the bacterium, Mycobacterium tuberculosis (Mtb), and is a leading cause of mortality from infectious disease. Mtb is an opportunistic pathogen that benefits from a compromised immune system and can lead to a statistically increased risk of HIV/TB co-infection, which leads to a significant increase in the mortality rate of HIV patients. Treatment has become difficult due to an increase in multi-drug resistant strains. Our collaborators at Texas Southern University have identified novel bacterial methionine aminopeptidase (MtMetAP1) inhibitors. Genetic knockout studies support the lethality of the deletion of MtMetAP1 in Mtb. Further studies support the existence of an ortholog of MtMetAP1 in Homo sapiens (HsMetAP1). Humans have several paralogs of MetAP, which perform similar essential functions to MetAP1. Although these novel inhibitors are known to be effective against Mtb bacteria, their cytotoxicity in mammalian cells has yet to be established. We hypothesize that there will be low cytotoxicity in cancerous and primary lung cell lines at varying concentrations of the MetAP novel inhibitor, UST-001. Using flow cytometry via a MUSE Cell Count and Viability Kit, the cytotoxicity of UST-001 was tested in H1299 human lung carcinoma cells. The H1299 cells were exposed for 48 hours to concentrations of UST-001 ranging from 1μM to 75μM. Three controls were included in each replicate: DMSO (carrier), pure media, and Isoniazid (INH), which is a common clinically effective TB treatment. When compared to the controls, even at the highest inhibitor concentration, 75 µM, the viability of H1299 cells was over 60%. Trypan Blue Assays were performed on the same H1299 cells for each concentration and controls which corroborated flow results. In establishment of a dose-response curve, our data showed that at UST-001 concentrations below 10 μM, there was little to no cell death and a greater than 90% viability. To see a significant decrease in percent viability, albeit to only 60%, the concentrations had to be increased to 75μM. We are currently establishing the cytotoxicity of UST-001 in healthy non-cancerous cells, with a second cell line, Mlg-2908, a non-cancerous mouse lung fibroblast line. Since cancerous cells have higher mitotic rates and more repair pathways, we hypothesize that 1) we will be able to establish anIC50 and 2) it will be closer to 10 μM in non-cancerous cells. Further studies using a Muse Annexin V apoptosis assay will focus on the UST-001 mechanism of cell death. Our ultimate goal is to characterize the toxicity of this drug in mammalian systems leading to its potential of being a new class of antibiotics and ultimately its promise of yet a new drug target.